Prion diseases are fatal neurodegenerative disorders of humans and animals. They result from conversion of PrPC, a normal membrane glycoprotein into PrPSc, a conformationally altered isoform that is infectious in the absence of nucleic acid. Most exogenously acquired prion diseases arise by exposure to the infectious agent outside of the central nervous system (CNS). For this reason, a major focus of research in the field has been to understand how prions gain access to the CNS from the periphery, and how they spread within the spinalcord and brain. To facilitate studies of prion trafficking, we propose to utilize PrP molecules fused to enhanced green fluorescent protein (EGFP). Such PrP-EGFP fusion proteins make it possible to analyze the cellular trafficking of PrPSc in living cells in real time, and to avoid the artifacts associated with conventional immunocytochemical detection of PrPSc. We previously created Tg(PrP-EGFP) mice in which EGFP was inserted adjacent to the glycolipid attachment site of PrP. This form of PrP-EGFP serves a highly specific ligand that binds to and labels intracellular and extracellular deposits of PrPSc in prion-infected animals. However, this particular fusion protein is not itself converted into PrPSc-EGFP, and so is subject to several experimental limitations. In this two-year project, we propose to design improved PrP-EFGP constructs that can be converted efficiently into PrPSc-EGFP. We will then use these fluorescent proteins to visualize the transport of PrPSc along the axons of living neurons to determine whether this movement is an intra-axonal, motor-driven process, or occurs via a "domino mechanism" on the axolemma. We expect that the proposed experiments will provide important insights into how prions spread along nerves,and suggest how this process can be manipulated as a therapeutic or prophylactic strategy.